Project Summary/Abstract Catalytic, site-selective C-H bond functionalization has enormous potential to simplify the synthesis of biologically relevant molecules by replacing complex, synthetic sequences starting with single-step functionalizations of C-H bonds at positions that are typically inert. This simplification could eliminate the need for substrates with specifically placed functionality, improve atom economy, and enable the late-stage diversification of medicinally active compounds. The ubiquity of C-H bonds in biological molecules provides chemists multiple opportunities for diversification of these structures, but concomitant challenges from the need to functionalize a specific C-H bond over other equally reactive C-H bonds. This goal of site-selective C-H bond functionalization is being sought predominantly with small molecule catalysts. One strategy to overcome these challenges is to combine Nature?s molecular recognition with chemists? reactivity to create artificial metalloenzymes that combine the exquisite selectivity and specificity of an evolvable protein scaffold with the reactivity of synthetic transition metal complexes. Several bioengineered artificial metalloenzymes catalyze abiological transformations, including those based on heme proteins. However, groundbreaking research published in Nature and Science from the Hartwig laboratory, in collaboration with Prof. Doug Clark, showed that replacing the iron in the porphyrin IX framework with noble metals led to discrete reconstitu ted proteins that catalyze the enantioselective insertions of carbenes into C-H bonds, a reaction class that had not been catalyzed by either natural or artificial enzymes previously. Subsequent studies on C-H amination with similar cytochrome P450 enzymes containing noble-metal cofactors led to the intramolecular amination of a C-H bond with greater than 25:1 chemoselectivity for C-H insertion over reduction of the azide reagent. Although the selectivity of the developed noble-metal artificial metalloenzyme is excellent, the amination process is limited to examples of intramolecular reactions, and the scope of the reactions are confined to group-transfer processes because of the primary coordination sphere of the porphyrin IX substructure. When the PI starts his research in Berkeley in September 2017, he will conduct studies to establish methods to create new artificial metalloenzymes that will have the potential to catalyze the intra- and inter-molecular amination of C-H bonds with a broader scope. To do so, the PI will incorporate into cytochrome P450 proteins modified porphyrins and salens to increase the reactivity of the intermediates in C-H bond amination, as well as planar, tridentate cofactors that allow coordination geometries beyond those enforced by planar tetradentate ligands. Accomplishing this goal will greatly increase the scope of synthetic transformations catalyzed by artificial metalloenzymes, and such discoveries will open new avenues for the generation and exploration of pharmacological products and lay the foundation to use artificial metalloenzymes in new synthetic contexts.